The use of antibodies as therapeutic treatment for a variety of diseases and disorders are rapidly increasing because they have shown to be safe and efficacious therapeutic agents. Approved therapeutic monoclonal antibodies for human use include Trastuzumab (antigen: HER2/neu), Edrecolomab (antigen: Ep-CAM), Anti-human milk fat globules (HMFG1) (antigen: HMW Mucin), Cetuximab (antigens: EGF receptor), Alemtuzumab (antigen: CD52), and Rituximab (antigen: CD20). Additional monoclonal antibodies are in various phases of clinical development for humans for a variety of diseases with the majority targeting various forms of cancer.
Antibodies target an antigen through its binding of a specific epitope on an antigen by the interaction with the variable region of the antibody molecule. Furthermore, antibodies have the ability to mediate and/or initiate a variety of biological activities. For example, antibodies can modulate receptor-ligand interactions as agonists or antagonists. Antibody binding can initiate intracellular signalling to stimulate cell growth, cytokine production, or apoptosis. Antibodies can deliver agents bound to the Fc region to specific sites. Antibodies also elicit antibody-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and phagocytosis.
While the properties of antibodies make them very attractive therapeutic agents, there are a number of limitations. There are several methods being utilized to generate antibodies including hybridoma technology, ribosome display, bacterial and yeast display, and others known in the art. The vast majority of monoclonal antibodies (mAbs) are of rodent origin. When such antibodies are administered in a different species, patients can mount their own antibody response to such xenogenic antibodies. Such response may result in the eventual neutralization and elimination of the antibody. One solution to this challenge involves the process of engineering an antibody with sequences compatible with the species subjected to the treatment. This process can prevent or greatly delay the patient developing an immune response against the administered therapeutic monoclonal antibody and extends the half-life of that antibody in the circulation of the treated subject. Such approaches, however, require careful balancing so that the antibody retains specificity and binding.
These limitations have prompted the development of engineering technologies known as “humanization”. Humanized antibodies can be generated as chimeric antibodies or fragments thereof which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human antibodies (i.e. “recipient antibody” or “target species antibody”) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (i.e. “donor antibody” or “originating species antibody”) such as mouse, having the desired properties such as specificity, affinity, and potency. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. This humanization strategy is referred to as “CDR grafting” as reported for the making of humanized antibodies (Winter, U.S. Pat. No. 5,225,539). Back mutation of selected target framework residues to the corresponding donor residues might be required to restore and/or improved affinity. Structure-based methods may also be employed for humanization and affinity maturation, for example as described for humanization in U.S. patent application Ser. No. 10/153,159 and related applications. Comparison of the essential framework residues required in humanization of several antibodies, as well as computer modeling based on antibody crystal structures revealed a set of framework residues termed as “Vernier zone residues” (Foote, J. Mol. Biol. 224:487-499 (1992)). In addition, several residues in the VH-VL interface zone have been suggested to be important in maintaining affinity for the antigen (Santos, Prog Nucleic Acid Res Mol. Biol. 60:169-94 (1998); Kettleborough, et al., Protein Engin., 4:773-783 (1991)). Similar strategies for “caninization” of antibodies for use in dogs are described in WO 03/060080.
Alternatively, humanized antibodies may contain the CDRs from a non-human sequence grafted into pools (e.g. libraries) of individual human framework regions. This newly engineered antibody is able to bind to the same antigen as the original antibody. The antibody constant region is derived from a human antibody. The methodology for performing this aspect is generally described as framework shuffling (Dall'Acqua, Methods, 36:43-60 (2005)). Furthermore, the humanized antibody may contain sequences from two or more framework regions derived from at least two human antibody germline sequences with high homology to the donor species. Antibodies designed using this method are described as hybrid antibodies (Rother et al., U.S. Pat. No. 7,393,648).
The approaches described above utilize essentially entire framework regions from one or more antibody variable heavy chains or variable light chains of the target species which are engineered to receive CDRs from the donor species. In some cases, back mutation of selected residues in the variable region is used to enhance presentation of the CDRs. Designing antibodies that minimize immunogenic reaction in a subject to non-native sequences in the antibody, while at the same time preserving antigen binding regions of the antibody sufficiently to maintain efficacy, has proven challenging.
Another challenge for developing therapeutic antibodies targeting proteins is that epitopes on the homologous protein in a different species are frequently different, and the potential for cross-reactivity with other proteins is also different. As a consequence, antibodies have to be made, tested and developed for the specific target in the particular species to be treated.